Transgenic corn with antifungal peptide AGM182 (DN:0113.18)

ABSTRACT

Aspergillus flavus  is an opportunistic, saprophytic fungus that infects maize and other fatty acid-rich food and feed crops and produces toxic and carcinogenic secondary metabolites known as aflatoxins. In vitro studies showed a five-fold increase in antifungal activity of AGM182 (vs. tachyplesin1) against  A. flavus . Transgenic maize plants expressing AGM182 under maize Ubiquitin-1 promoter were produced through  Agrobacterium -mediated transformation. PCR products confirmed integration of the AGM182 gene, while RT-PCR of maize RNA confirmed the presence of AGM182 transcripts. Maize kernel screening assay using a highly aflatoxigenic  A. flavus  strain (AF70) showed up to 72% reduction in fungal growth in the transgenic AGM182 seeds compared to isogenic negative control seeds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/134,336, filed Sep. 18, 2018, the entire contents of which areincorporated herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application includes an electronically submitted sequence listingin .txt format. The .txt file contains a sequence listing entitled“2018-10-29 NEXION ST25.txt” created on Oct. 29, 2018 and is 2,985 bytesin size. The sequence listing contained in this .txt file is part of thespecification and is hereby incorporated by reference herein in itsentirety.

FIELD OF THE INVENTION

Control of Aspergillus flavus growth and aflatoxin production intransgenic maize kernels expressing a tachyplesin-derived syntheticpeptide, AGM182.

BACKGROUND OF THE INVENTION

Aspergillus flavus is an opportunistic, saprophytic fungus that infectsfatty acid-rich food and feed crops including maize [1]. Upon infection,the fungus produces toxic and carcinogenic secondary metabolites knownas aflatoxins. Aflatoxin contamination of maize has an economic impactof approximately $163 million/year in the U.S. [2]. With rapid change inglobal climate, it is predicted that aflatoxin contamination could causelosses to the maize industry up to US$1.68 billion per year in theUnited States [3]. Besides crop loss, the adverse effect of aflatoxinson human and animal health is acute [1] that can result in liver cancer.Different approaches are being explored to alter genetically, througheither traditional breeding efforts or other gene manipulationtechniques, maize varieties for increased resistance to preharvestaflatoxin contamination [4]. Previously published reports indicate theusefulness of naturally occurring antimicrobial peptides (AMPs), alsoknown as host defense peptides, such as cecropin, melittin, magainin,defensin and protegrin for controlling a variety of phytopathogens[5-9].

AMPs are evolutionarily highly conserved components of the innate immunesystem, which provide the first line of defense against invadingpathogens [10]. Their significance in host defense is underscored inplants and insects as these can live in bacterial environments withoutthe ability to produce lymphocytes and antibodies. AMPs have directantimicrobial activities and kill Gram-negative and -positive bacteria,as well as fungi and protozoa [11-14]. They often assume secondarystructures in the form of amphipathic α-helices or partial β-pleatedsheets that latter being ‘conformationally-locked’ by disulfidelinkages. AMPs exert biological activity by interactingelectrostatically and perturbs the pathogen's membrane thereby impairingits function as a barrier [15]. Ergosterol, the membrane sterol foundalmost exclusively in fungi and present in conidial walls is a highlyselective target of AMPs and this interaction leads to lytic membranedisruption [16]. Mammalian cells, which contain more zwitterionicphospholipids framed with cholesterol and cholesterol esters, are muchless disturbed by AMP interactions. This targeting effect and directcontact disruption of the pathogen's membrane makes resistance lesslikely to develop [17]. Unfortunately, natural peptides are subject torapid degradation in the cytoplasm reducing their effectiveness inplanta. Designed antimicrobial peptides (dAMPs) arelaboratory-synthesized peptides that have been rationally and chemicallydesigned from naturally occurring AMPs. Based upon the functions andstructure in naturally occurring AMPs, structural algorithms have beendeveloped that have added numerous variations to the structuralrepertoire by design, laboratory created peptides have demonstratedincreased potency, efficacy, safety, specificity and reduced toxicity incomparison to their natural templates [7, 18]. Synthetic peptides, whichare fairly resistant to cytoplasmic degradation [16], are useful incontrolling a broad-spectrum of plant pathogens including thesaprophytic fungus, A. flavus [19, 20]. The effectiveness of syntheticpeptides such as D4E1, D2A21 or MS199 expressed in transgenic cotton andother crops for controlling Aspergillus and other microbial pathogenshas been demonstrated in our laboratory and elsewhere (see [21]).Synthetic peptides, which can be designed to be fairly resistant tocytoplasmic degradation, are also effective in controlling abroad-spectrum of microbial plant pathogens including themycotoxin-producing fungal species—Aspergillus and Fusarium. Thesepeptides also exhibit no toxicity against mammals and non-targetbeneficial organisms [22, 23]. We had documented earlier theeffectiveness of a cecropin-based synthetic, lytic peptide, D4E1, incontrolling A. flavus infection in transgenic cottonseed in situ and inplanta [20]. Schubert et al. [24] showed partial resistance againstAspergillus in transgenic maize expressing a recombinant thanatinpeptide. We have recently evaluated another group of synthetic peptides,modelled after tachyplesin1—a defensin-like peptide [25], found in theacid extract of hemocytes from the Japanese horseshoe crab (Tachypleustridentatus), against

flavus [26]. In this report, we describe the design and synthesis of atachyplesin-based antimicrobial peptide AGM182. We tested its efficacyin vitro to inhibit A. flavus growth as compared to the nativetachyplesin1. We also produced transgenic maize lines expressing thesynthetic peptide AGM182 and assayed the seeds for anti-A. flavusactivity and determined aflatoxin levels in transgenic lines. Reductionof aflatoxin contamination through transgenic expression of a syntheticpeptide in an important food crop such as maize is detailed in thisreport. The results presented here show the effectiveness ofcomputational and synthetic biology to rationally design, synthesize,and validate an AMP against A. flavus that is effective in reducingfungal growth and aflatoxin contamination in a major crop like maize.

BRIEF DESCRIPTION OF THE INVENTION

Aspergillus flavus is an opportunistic, saprophytic fungus that infectsmaize and other fatty acid-rich food and feed crops and produces toxicand carcinogenic secondary metabolites known as aflatoxins.Contamination of maize with aflatoxin poses a serious threat to humanhealth in addition to reducing the crop value leading to a substantialeconomic loss. Here we report designing a tachyplesin1-derived syntheticpeptide AGM182 and testing its antifungal activity both in vitro and inplanta. In vitro studies showed a five-fold increase in antifungalactivity of AGM182 (vs. tachyplesin1) against A. flavus. Transgenicmaize plants expressing AGM182 under maize Ubiquitin-1 promoter wereproduced through Agrobacterium-mediated transformation. PCR productsconfirmed integration of the AGM182 gene, while RT-PCR of maize RNAconfirmed the presence of AGM182 transcripts. Maize kernel screeningassay using a highly aflatoxigenic A. flavus strain (AF70) showed up to72% reduction in fungal growth in the transgenic AGM182 seeds comparedto isogenic negative control seeds. Reduced fungal growth in the AGM182transgenic seeds resulted in a significant reduction in aflatoxin levels(76-98%). The results presented here show the power of computational andsynthetic biology to rationally design and synthesize an antimicrobialpeptide against A. flavus that is effective in reducing fungal growthand aflatoxin contamination in an economically important food and feedcrop such as maize.

More specifically, the present invention is referred to a method toproduce transgenic maize lines expressing synthetic peptide, AGM182, themethod comprising the steps of:

a. designing a gene AGM182 comprising 129 bp fragment from naturallyoccurring antimicrobial peptide tachyplesin 1, the designing comprising:[0009] b. eliminating second disulfide linkage of peptide tachyplesin 1;and [0010] c. replacing a sequence from the peptide tachyplesin 1 byamino acid residues that results in amphipathic β-sheet conformationwith higher positive charge density, higher hydrophobicity, anti-fungalactivity and stability; [0011] d. cloning the AGM182 gene into a pMCG1005 plasmid under a constitutive Ubi-1 (maize) promoter; [0012] e.incorporating a maize alcohol dehydrogenase-1 (Adh-1) intron into theupstream of the AGM182 start codon, wherein the Adh-1 intron improvesthe expression of the AGM182 gene; [0013] f fusing a barley alphaamylase signal peptide (BAAS) to the N-terminal end of the AGM182 gene,wherein the BAAS peptide increases the efficiency of AGM182 excretionfrom the host cell to the cell wall; [0014] g. producing the transgenicmaize lines through an Agrobacterium EHA101-mediated transformation ofthe AGM182 gene into immature maize embryos.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (SEQ ID NOS: 1 and 2) is a schematic diagram showing the physicalproperties of both peptides compared by casting the single letter aminoacid codes (top row) into Molly (bottom row) [36, 42], a glyph-baseddesign tool.

FIGS. 2A, 2B shows three dimensional depictions of the tachyplesin1(FIG. 2A, SEQ ID NO: 2) and AGM182 (FIG. 2B, SEQ ID NO: 1) weregenerated in PyMol, a molecular visualization system maintained anddistributed by Schrodinger, a computational chemistry company(Cambridge, Mass.).

FIGS. 3A to 3C show AGM182 gene and vector information. FIG. 3A showsthe AGM182 nucleotide sequence (SEQ ID NO:3), FIG. 3B shows the aminoacid sequence (SEQ ID NO:4), and FIG. 3C is a pMCG1005 vector diagramcontaining AGM182 expression cassette used for maize transformationusing the Agrobacterium strain.

FIG. 4 is a graph that shows the inhibitory activity in vitro oftachyplesin1 and tachyplesin-based synthetic peptide, AGM182 againstpre-germinated conidia of A. flavus. AGM182 showed a better inhibitoryactivity (IC₅₀=2.5 μM) than tachyplesin1 against A. flavus (IC₅₀=12.5μM). Average means of three independently replicated assays.

FIGS. 5A and 5B. Molecular analyses of AGM182 expressing transgenicmaize plants. (FIG. 5A) PCR confirmation of transgenic plants containingAGM182 gene (129 bp diagnostic DNA fragment; +ve=plasmid control;C=isogenic negative control; (FIG. 5B) AGM182 expression (90 bp product)in the seeds of transgenic lines [vs. negative control (C)] usingRT-PCR. Maize ribosomal structural gene (Rib), GRMZM2G024838 [33], wasused as house-keeping gene. (132 bp product). NEB 2 log DNA ladder wasused as a DNA marker.

FIG. 6 shows GFP fluorescence emanating from the A. flavus strain 70-GFPin transgenic kernels after one week of bioassay. Isogenic negativecontrol kernels (neg) were used to evaluate the fungal infection andspread in transgenic kernels (Lines 2-5). Up to 10 kernels were screenedfor each line under the fluorescent microscope and a representativekernel is shown here.

FIGS. 7A and 7B show A. flavus infection, growth, and aflatoxinproduction after 7 d in a Kernel Screening Assay. FIG. 7A shows theaverage of 12 biological replicates and each replicate contained atleast four kernels. GFP quantification (in Relative Fluorescence Units)which indicates fungal growth and FIG. 7B shows aflatoxin levels intransgenic A. flavus-infected T₃ maize kernels (Lines 1-6 as comparedwith an isogenic negative control). Mean separation was done byDunnett's posttest after ANOVA. Levels of significant reduction isdenoted by asterisks (**=95%; ***=99% probability levels). Error barsindicate standard error of means for four randomized biologicalreplicates.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 (SEQ ID NOS: 1 and 2) shows the physical properties of bothpeptides compared by casting the single letter amino acid codes (toprow) into Molly (bottom row) [36, 42], a glyph-based design tool. Cyancolor represents those amino acids that are hydrophobic while those thatare hydrophilic retain a magenta color. Disulfide linkages are denotedby the green lines connecting the relevant cysteines, which are yellowin color. While a similar ‘central bubble’ has been maintained in theAGM182 (CLGKFC)(SEQ ID NO: 5) compared to that of tachyplesin1(CYRGIC)(SEQ ID NO: 6), the second disulfide linkage of tachyplesin1 hasbeen eliminated in AGM182 and replaced by a sequence that assumes anamphipathic beta-sheet conformation with maximized positive chargedensity. This property has been demonstrated to enhance anti-fungalactivity of dAMP peptides [19, 36]. The red dots on AGM182 denote thedifferences in amino acid sequence compared to tachyplesin1.

The main highlights of the present invention can be summarized as:

Designed the synthetic peptide AGM182 modeled after the naturallyoccurring tachyplesin 1.

AGM182 was five-times more effective in controlling Aspergillus flavuscompared to tachyplesin 1.

Transgenic maize plants expressing the synthetic peptide AGM182 wereproduced and advanced to third generation by selfing.

Kernel Screening Assay showed significant reduction in fungal growth(72%) and spread inside transgenic kernels.

Concomitant, significant reduction in aflatoxin levels (76-98%) was alsoachieved in transgenic kernels.

The abbreviations to be used in the present specification are: RB=rightborder, LB=left border, Ubi-1=maize Ubiquitin) promoter (constitutive),Int=intron (maize alcohol dehydrogenase-1; Adh1), AGM182=gene ofinterest; BAAS=barley alpha amylase signal peptide, ocs (octopinesynthase)=terminator, nos (nopaline synthase)=terminator,4×35S=constitutive cauliflower mosaic virus promoter,Bar=phosphinothricin acetyltransferase gene for BASTA herbicideresistance.

Materials and Methods

In Silico Analysis of AGM182

The synthetic peptide AGM182 used in the current study were designedbased on the known antimicrobial peptide tachyplesin1 (FIGS. 1 and 2 ).The 3D structures of the AMPs were generated using PyMol software,maintained and distributed by Schrodinger, a computational chemistrycompany (Cambridge, Mass.). The AMPs were analyzed using the AMPpredictor tools, ‘Antimicrobial Peptide Calculator and Predictor’(Antimicrobial Peptide Database with APD3 algorithm; aps.unmc.edu/AP/[27]. Tachyplesin1 was synthesized by Bachem (Bubendorf, Switzerland)and AGM182 synthetic peptide (AgroMed/Nexion Biosciences Ltd, Riva, Md.)was synthesized by Biomatik (Cambridge, Ont, Canada), both with a purityof >95% as obtained by the analyses of HPLC and Mass Spectrometry dataprovided by the manufacturers.

Plasmid Constructs and Maize Transformation

A 129 bp fragment of the AGM182 gene (SEQ ID NO:3) was synthesized (IDT;Coralville, Iowa) and cloned in to the pMCG 1005 vector [28] (providedby Dr. Kan Wang, IA State University). The synthetic AGM182 gene (codonoptimized for expression in maize) was expressed under the constitutiveUbi-1 (maize) promoter present in the plant destination vector. Themaize alcohol dehydrogenase-1 (Adh1) intron present in the transgenecassette (upstream of the AGM182 start codon) was incorporated toimprove the expression of AGM182 in maize (monocot) and the barley alphaamylase signal peptide (BAAS) was fused to the N-terminal end of theAGM182 gene (FIG. 3C) to increase the efficiency of AGM182 (peptide)excretion from the host cells to the cell wall [29]. AgrobacteriumEHA101-mediated transformation of immature maize (Zea mays L. Hi-II)embryos was accomplished through the Plant Transformation Facility atthe Iowa State University [30]. Maize seedlings were grown at 25° C.under 16 h photoperiod (80).

μmol m-2 s-1) in a growth chamber for 4 weeks and then moved in to thegreenhouse in 5-gallon (19 L) pots [31]. Transgenic plants fromindependently transformed events were grown in moist soil mix containing3 parts Scott's 360 Metro-Mix (Scotts Company, Marysville, Ohio) and 1part perlite in 7.5 cm pots and were selfed to obtain T₃ generationkernels. Isogenic maize lines that went through transformation processbut tested negative by PCR and herbicide assay [31] were used asnegative controls.

Fungal Strain and Bioassay with Peptides

Aspergillus flavus 70-GFP [32] was grown at 31° C. on V8 medium (5% V8juice, 2% agar, pH 5.2). Spores from 6-day old cultures were suspendedin 0.02% Triton X-100; the conidial concentration was determined with ahemocytometer and adjusted to 4×10⁶ conidia ml-1.

Peptides were freshly dissolved in sterilized water and used forantifungal bioassays as reported [19]. Briefly, pre-germinated conidialsuspensions (4×10⁶ conidia ml⁻¹) of A. flavus 70-GFP were treated withthe peptides at 0-25 μM concentrations for 60 min before spreading onPotato Dextrose Agar plates (9 cm day). Fungal colonies were enumeratedfollowing incubation at 30° C. for 24 h.

PCR Screening of Transgenic Maize Kernels

Maize seeds were flash frozen and ground using a 2010 Geno/Grinder (SPEXSamplePrep, Metuchen, N.J.). Transgenic plants were screened through PCRusing ‘Phire Plant Direct PCR Kit’ (ThermoFisher Scientific, Waltham,Mass.) according to the manufacturer's protocol. The screening primersused in this study were, AGM182_F1: 5′-ATGGCCAACAAGCATCTGTC-3′ (SEQ IDNO: 7) and AGM182_R1: 5′-CCGCGCCTTTATACAGAACT-3′ (SEQ ID NO: 8). A 51°C. annealing temperature and 10 s elongation time were used to amplify a129 bp DNA fragment to confirm the presence of the AGM182 gene in thetransgenic maize plants.

RNA Isolation, cDNA Synthesis, and Semi-Quantitative RT-PCR

Pulverized maize seeds were used for RNA isolation using the Spectrum™Plant Total RNA kit′ (Sigma-Aldrich, St Louis, Mo.). cDNA wassynthesized using iScript™ cDNA synthesis kit (Bio-Rad, Hercules,Calif.) according to the manufacturer's protocol. Semi-quantitativeRT-PCR was performed using T100™ thermal cycler system (Bio-Rad) andPhusion® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich,Mass.). The thermocycling conditions included a

pre-incubation at 98° C. for 30 s followed by 30 cycles of 98° C. for 10s (denaturation), 51.5° C. for 30 s (primer annealing), 72° C. for 5 s(elongation), and a final extension step at 72° C. for 5 min. Theprimers used for RT-PCR of AGM182 in transgenic plants are qAGM182-F25′-TGGCCAACAAGCATCTGT-3′ SEQ ID NO: 9) and qAGM182-R25′-ACAGGCGCGCTTTAATCT-3′ (SEQ ID NO: 10) and maize ribosomal structuralgene (Rib), GRMZM2G024838 [33], qRib-F 5′-GGCTTGGCTTAAAGGAAGGT-3′ (SEQID NO: 11) and qRib-R 5′-TCAGTCCAACTTCCAGAATGG-3′ (SEQ ID NO: 12).

Kernel Screening Assay

Undamaged T₃ maize and negative control seeds were surface-sterilizedwith 70% ethanol and subjected to the Kernel Screening Assay (KSA) [32].Surface-sterilized seeds were briefly immersed in a 4×10⁶ conidialinoculum and incubated in the dark at 31° C. and high humidity (>95%RH). After seven days, four representative seeds were randomly chosenand photographed (bright field and fluorescence) using an Olympus SZH10research stereomicroscope equipped with the Nikon Digital CameraDXM1200.

GFP Fluorescence and Aflatoxin Analysis

To quantify fungal fluorescence pulverized A. flavus infected maizeseeds (50 mg FW) were extracted in 0.5 ml of Sorenson's phosphate buffer(pH 7.0). The samples were vortexed for 30 s followed by centrifugationat 10,000 g for 15 min. A 100 μl aliquot of the supernatant wastransferred to each well of a 96 well plate and GFP fluorescence(excitation 485 nm, emission

535) were recorded using a plate reader (Biotek Synergy 4, Winooski,Vt.) [32, 34]. Relative Fluorescence Units (RFU) were normalized aspercent values and used for statistical analysis from 12 biologicalreplicates. Each replicate consisted of four randomly selected, PCRpositive kernels. Following molecular analysis and GFP quantitation,seed samples were pooled together into four randomized replicates.Homogenates from three maize seeds were pooled, dried in a forced airoven (60° C.), and extracted with methylene chloride [35]. Sampleresidues were dissolved in 4.0 ml 80% methanol and total aflatoxinlevels were measured with the FluoroQuant Afla Test Kit for AflatoxinAnalysis (Romer Labs, Union, Mo.).

Statistical Analysis

All data from two independent KSAs including fluorescence (12 biologicalreplicates), and aflatoxin values (four randomized replicates) weresubjected to one-way ANOVA and mean separation was performed using theDunnett's posttest (P<0.05 or <0.01) using GraphPad Prism software (LaJolla, Calif.).

Results

Design and in Silico Analysis of AGM182

The synthetic AGM182 peptide was designed based on the naturallyoccurring Tachyplesin I peptide from Japanese horseshoe crab. Analysisof amino acids in the AGM182 shows increase in positive charge density(vs. Tachyplesin1). While a similar ‘central bubble’ (CLGKFC)(SEQ ID NO:5) has been maintained in the AGM182 compared to that of Tachyplesin1(CYRGIC)(SEQ ID NO: 6), the second disulfide linkage of Tachyplesin1 waseliminated in AGM182 and replaced by a sequence that results into anamphipathic β-sheet structure conformation with maximized positivecharge density for improved antifungal activity FIG. 1 . Thethree-dimensional structures of both Tachyplesin I and AGM-182 are shownin FIG. 2A (tachyplesin1 (SEQ ID NO: 2) and FIG. 2B (AGM182 (SEQ ID NO:1)). The physical-chemical properties of both AMPs, that include lengthof the peptide, amino acid composition, charge, molecular weight,hydrophobicity, and Boman index. Hydrophobicity was significantlyincreased in the AGM182 peptide.

In Vitro Testing of Antimicrobial Activity of the Peptides

The synthetic peptide AGM182 was evaluated for activity against A.flavus in comparison to the native peptide tachyplesin1. AGM182 showedfivefold increase in its IC₅₀ value against A. flavus as compared totachyplesin1 (IC₅₀=2.5 μM vs. 12.5 μM; FIG. 3C). No hemolysis of porcineblood cells was observed at all concentrations of AGM182 compared to100% hemolysis with 0.1% Triton-X.

Maize Transformation and Molecular Screening of Transgenic Plants

Transformation of maize (Hybrid Hi-II) was accomplished using theAgrobacterium tumefaciens EHA101-mediated transformation of immatureembryos [31]. The codon-optimized synthetic AGM182 gene was expressedunder the constitutive Ubi-1 promoter FIG. 3C. Thirteen independentlytransformed TO lines were generated and eleven T₁ lines were foundpositive for the presence of both the herbicide marker gene and AGM182.Eight T₂ lines were selected and advanced to T₃ generation in thegreenhouse by selfing and only six lines expressing the AGM182 geneprovided sufficient number of kernels for further assays. Transgenicmaize plants did not exhibit any overt phenotype compared to isogenicnegative or non-transformed control plants (data not shown). PCRscreening of the transgenic maize seed showed the presence of 129 bpamplicon (AGM182 specific) in the AGM182 transgenic plants that wasabsent in the control plants (FIG. 5A). Expression analysis of theAGM182 transcripts in the AGM182 positive plants demonstratedsubstantial expression of this synthetic gene and no expression wasobserved in the control plants (FIG. 5A).

A. flavus Growth During Infection of Transgenic Seeds

A. flavus growth was monitored in transgenic seeds using the KSA.Expression of the AGM182 gene in transgenic maize kernels resulted in asignificant decrease in fungal growth as compared to the control (FIG. 6). GFP fluorescence, as an indicator of fungal growth inside the kernelembryo and/or endosperm positively correlated with the extent of fungalgrowth. AGM182-expressing maize kernels showed significant reduction infungal growth (up to 72%) as indicated by the decrease in GFPfluorescence compared to the isogenic control (FIG. 7A).

Aflatoxin Production in Transgenic Kernels

Transgenic expression of the AGM182 gene in maize significantly reducedaflatoxin content in the transgenic lines as compared to the control. Ingeneral, between the two aflatoxins (B₁ and B₂) primarily detected in A.flavus infected maize kernels, aflatoxin B₁ is the predominantaflatoxin. The aflatoxin data presented here is the total amount ofaflatoxins detected in the infected kernels. A significant reduction inaflatoxin levels (76-98%) was observed in the different AGM182 lines ascompared to an isogenic negative control (FIG. 7B bottom). Among the sixdifferent AGM182 lines studied here, transgenic Line 4 had the lowestaflatoxin level in the seeds (60.0 ng·g-1) as compared to the controlseeds (3000 ng·g-1).

Discussion

Designed synthetic peptides are effective against in controlling a broadspectrum of plant pathogens [5, 7, 9, 19, 36] includingdifficult-to-control, mycotoxin producing fungal species such asAspergillus and Fusarium. Their advantages over naturally occurringpeptides include increased resistance to proteolytic degradation [16,37, 38], no or minimal effect on off-target beneficial microbialpopulations [22, 23] and they can be integrated into genomes ofcultivated field, fruit and ornamental crops [20, 39-41] to provideresistance to diseases and toxin-producing fungal species. In thecurrent study the synthetic antimicrobial peptide AGM182 was designedbased on the naturally occurring AMP tachyplesin1 (FIG. 1 ).Physicochemical properties of AMPs play a significant role on theantimicrobial activity of AMPs. These include net positive charge/chargedensity, hydrophobicity and others [36]. Though the peptide length andnet positive charge in the AGM182 did not differ much with tachyplesin1,hydrophobicity was significantly increased in AGM182 (FIG. 1 ).Hydrophobicity is one of the important criteria for antimicrobialproperty of AMPs as it increases the interaction between AMPs and cellmembrane of target pathogens. Higher hydrophobicity in AMPs has beenshown to increase antimicrobial activity [36]. While designing AGM182, asimilar ‘central bubble’ though preserved in the AGM182 (CLGKFC)(SEQ IDNO: 5) vs. tachyplesin1 (CYRGIC)(SEQ ID NO: 6), the second disulfidelinkage was eliminated in AGM182 and replaced by amino acid residuesthat resulted in amphipathic β-sheet conformation with higher positivecharge density (FIG. 1 ). This positive attribute in dAMP peptides isshown to affect stability and anti-fungal activity [19, 42]. As such, afive-fold higher antimicrobial activity of AGM182 was observed ascompared to tachyplesin1 (IC₅₀=2.5 μM vs 12.5 μM) in vitro (FIG. 4 ) andit is attributable to the structural alteration described above. We havealso demonstrated the absence of hemolytic activity by this peptide aswell. Among different physicochemical properties that positively affecthemolytic activity of AMPs, presence of specific amino acid residuesespecially tryptophan, has been demonstrated to increase significantlylysis of mammalian erythrocytes by binding to cholesterol present inbiological membranes through the indole moiety [43]. Even exclusion ofonly tryptophan residue in melittin (an AMP from honeybee venom)significantly reduced its hemolytic activity [44]. The absence oftryptophan residue in AGM182 AMP could possibly explain its inability tolyse porcine erythrocytes.

The primary mode of action of these AMPs is membrane damage althoughAMPs have also been shown to be involved in cellular signaling and canmodulate host defense responses [45, 46].

Besides naturally occurring AMPs in plants, synthetic AMPs havedemonstrated the ability to restrict pathogen growth in vitro or inplanta (through transgenic approaches) [20, 39, 40, 47]. Earlier studiesfrom our lab in cotton showed transgenic expression of a synthetic AMP,D4E1, significantly reduced A. flavus growth and aflatoxin production incottonseed [20]. In this report we have demonstrated, prevention ofpreharvest aflatoxin contamination in a major food crop such as maizethrough transgenic expression of a tachyplesin-derived syntheticpeptide, AGM182.

In general, small peptides such as AGM182 used in the current study,could not be detected using the standard Western blotting technique inthe transgenic plants. Detection of such small antimicrobial peptides inplants are often challenging due to their small size (18 amino acids inAGM182, FIGS. 1 and 2 ) and lack of antigenicity to successfully raiseantibodies and this observation is in accordance with earlier reports onsynthetic peptides [18, 20, 24, 48, 49]. This also could be due to lowconcentration of the synthetic AGM182 peptide in transgenic maizekernels resulting from possible proteolytic degradation in the plants[16]; yet, the low AGM182 concentration in planta might still be goodenough to reduce A. flavus growth and subsequent aflatoxin production. Adirect correlation between in planta AGM182 production and fungal loadin the transgenic maize kernels could not be obtained due to theabove-mentioned reasons; however, at the transcript level fungal loadcorrelated well with the expression of AGM182 (FIG. 4 , FIGS. 5A-5B,FIG. 7A) in the transgenic kernels and corresponding decrease inaflatoxins (FIG. 7B).

The significant reduction in GFP fluorescence in the seeds of AGM182transgenic maize lines (FIG. 6 ) correlated with the reduction in fungalgrowth (FIG. 7A) and showed that AGM182 is functionally active in thetransgenic plants. Aflatoxin levels were drastically reduced (>95%) inthe infected AGM182 transgenic seeds (FIG. 7B), which is possibly due tothe reduction in fungal load and associated reduction in fungalpathogenicity during seed infection. Similar results were also observedin transgenic cotton seeds expressing the synthetic AMP, D4E1 [20]where, a significant reduction in A. flavus growth (45-70%) andaflatoxin production (75-98%) were observed, though the reduction inaflatoxin in the AGM182 transgenic maize seeds are relatively higherthan in the D4E1 transgenic cotton seeds. The higher reduction inaflatoxin contamination in maize by AGM182 (vs. D4E1 in cotton) could bedue to higher efficacy of AGM182 against A. flavus or could be a cropspecific response. At this stage, we do not know what other cellularimpact AGM182 might have on other metabolic processes in this fungus. Invitro on AGM182-A. flavus interaction using RNA-seq [50] in future mightreveal the impact of AGM182 on other metabolic processes in the fungusin addition to the known mode of action of AGM182 and other syntheticpeptides through destabilization of the fungal cell membrane leading tolysis [7, 19]. Any possible alteration in other metabolic pathways in A.flavus by AGM182 could possibly be an indirect effect resulting from theinstability of cellular structures caused by the AMP. Transgenicexpression of AGM182 in maize on the other hand, did not have anynegative effect on plant phenotype and seed weight in the transgenicplants as compared to the control suggesting that AGM182 does notinterfere with plant growth and associated yield attributes.

Overall, the results presented here demonstrated the effectiveness ofthe tachyplesin-derived synthetic peptide AGM182 on controlling A.flavus growth and aflatoxin contamination in transgenic maize kernels.In addition, this study highlights the potential application ofsynthetic biology to design efficiently a safe, synthetic AMP likeAGM182. The KSA results, as reported in this study (FIGS. 6 and 7A-7B),have been shown to be directly related to results obtained from fieldevaluations of corn genotypes [51]. It should be noted that even thebest performing transgenic maize lines in this screening recordedaflatoxin levels 60-150 ngg-1 under the optimal conditions. A single,highly contaminated kernel can produce a high aflatoxin value for theentire seed batch. Food and feed safety standards set forth by theEuropean Union, the USA and other countries limit total aflatoxin levelsto 4-20 ng·g-1[52]. It is noteworthy that the experimental conditionsemployed for the kernel screening assay were highly conducive forsuccessful colonization and toxin production by the fungus, which mightnot be the case under field environments. However, it appears that nosingle technology can completely inhibit A. flavus infection and reduceaflatoxin contamination to allowable levels imposed by regulations setfor food and feed commodities [53]. Thus, a combination of technologiessuch as use of resistant lines bred through classical or molecularapproaches [51, 54-57], application of atoxigenic strains to replacetoxin-producing strains [58], and good agronomic practices combined withpest control [4, 59] should assist in safeguarding human and livestockfrom dangerous levels of aflatoxins in contaminated food and feed.Conspicuously, much of the adverse health impacts due toaflatoxin-contaminated food and feed crops in developing countriesresults from consumption of improperly stored produce; hence, it isessential to explore avenues for control of aflatoxin contaminationduring storage and handling as well.

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What is claimed is:
 1. A method to produce transgenic maize linesexpressing a synthetic AGM182 peptide, the method comprising the stepsof: producing a transgenic maize line through an agrobacterium mediatedtransformation of the AGM182 gene into immature maize embryos, whereinthe AGM182 gene comprises a 129 bp nucleic acid of SEQ ID NO:3.
 2. Themethod as claimed in claim 1, wherein the nucleic acid of SEQ ID NO:3encodes an amino acid sequence of the AGM182 peptide of SEQ ID NO:
 4. 3.The method as claimed in claim 1, wherein the AGM182 peptide iseffective against mycotoxin producing fungal species and with notoxicity against mammals.
 4. The method as claimed in claim 1, whereinthe AGM182 peptide reduces the growth of the mycotoxin producing fungalspecies Aspergillus Flavus.
 5. The method as claimed in claim 1, whereinthe AGM182 peptide reduces the growth of Aspergillus Flavus by 72% intransgenic maize lines.
 6. The method as claimed in claim 1, wherein theAGM182 peptide reduces the growth of mycotoxin producing fungal species,wherein the fungal species is Fusarium.
 7. The method as claimed inclaim 3, wherein the mycotoxin is aflatoxin.
 8. The method as claimed inclaim 1, wherein the AGM182 peptide reduces an aflatoxin contaminationby 76-98% in transgenic maize lines.
 9. A transgenic maize lineexpressing an AGM182 peptide of SEQ ID NO:4 that reduces aflatoxinproduction and fungal growth of Aspergillus Flavus.
 10. The transgenicmaize line as claimed in claim 9, wherein the synthetic AGM182 peptide,reduces the growth of the Aspergillus Flavus by 72%.
 11. The transgenicmaize line as claimed in claim 9, wherein the synthetic AGM182 peptidereduces the aflatoxin contamination by 76-98%.
 12. An antimicrobialpeptide AGM182 of SEQ ID NO:4.
 13. The antimicrobial peptide as claimedin claim 12, wherein the synthetic antimicrobial AGM182 peptide has afive-fold higher antimicrobial activity (IC50=2.5 μM) compared tonaturally occurring antimicrobial peptide tachyplesin 1 (IC50=12.5 μM).14. A vector comprising an AGM182 gene that comprises a 129 bp nucleicacid of SEQ ID NO:3.
 15. The vector of claim 14, wherein the vector isan expression vector.
 16. The vector of claim 14, wherein the vector isa pMCG 1005 plasmid and the AGM182 gene is operatively linked to aconstitutive Ubi-1 (maize) promoter.
 17. The vector of claim 14, whereinthe vector further comprises a maize alcohol dehydrogenase-1 (Adh-1)intron that is 5′ from the AGM182 gene start codon, wherein the Adh-1intron improves the expression of the AGM182 gene.
 18. The vector ofclaim 14, wherein the vector further comprises a barley alpha amylasesignal peptide (BAAS) nucleic acid sequence that is 5′ from the AGM182gene, wherein the BAAS peptide increases the efficiency of the syntheticAGM182 peptide excretion from the host cell to the cell wall.
 19. Amethod of preventing or treating a plant to reduce a growth of amycotoxin producing fungal species comprising: spraying a seed,seedling, or plant with an AGM182 peptide of SEQ ID NO: 4 in an amountsufficient to prevent or treat the growth of the mycotoxin producingfungal species.